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Introduction Mayer-Rokitansky-Kuster-Hauser syndrome (MRKHS) is characterized by congenital absence of the uterus and the upper two-thirds of the vagina in otherwise phenotypically normal females. It is found isolated or associated with renal, skeletal and other malformations. Despite ongoing research, the etiology is mainly unknown. For a long time, the hypothesis of deficient hormone receptors as the cause for MRKHS has existed, supported by previous findings of our group. The aim of the present study was to identify unknown genetic causes for MRKHS and to compare them with data banks including a review of the literature. Material and methods DNA sequence analysis of the oxytocin receptor (OXTR) and estrogen receptor-1 gene (ESR1) was performed in a group of 93 clinically well-defined patients with uterovaginal aplasia (68 with the isolated form and 25 with associated malformations). Results In total, we detected three OXTR variants in 18 MRKHS patients with one leading to a missense mutation, and six ESR1 variants in 21 MRKHS patients, two of these causing amino acid changes and therefore potentially disease. Conclusions The identified variants on DNA level might impair receptor function through different molecular mechanisms. Mutations of ESR1 and OXTR are associated with MRKHS. Thus, we consider these genes potential candidates associated with the manifestation of MRKHS.

The BRAFV600E inhibitor vemurafenib achieves remarkable clinical responses in patients with BRAF-mutant melanoma, but its effects are limited by the onset of drug resistance. In the case of resistance, chemotherapy can still be applied as second line therapy. However, it yields low response rates and strategies are urgently needed to potentiate its effects. In a previous study, we showed that the inhibition of the PI3K-AKT-mTOR pathway significantly increases sensitivity of melanoma cells to chemotherapeutic drugs (J. Invest. Dermatol. 2009, 129, 1500). In this study, the combination of the mTOR inhibitor temsirolimus with the chemotherapeutic agent temozolomide significantly increases growth inhibition and apoptosis in melanoma cells compared to temsirolimus or temozolomide alone. The combination of temozolomide with temsirolimus is not only effective in established but also in newly isolated and vemurafenib-resistant metastatic melanoma cell lines. These effects are associated with the downregulation of the anti-apoptotic protein Mcl-1 and the upregulation of the Wnt antagonist Dickkopf homologue 1 (DKK1). Knock-down of DKK1 suppresses apoptosis induction by the combination of temsirolimus and temozolomide. These data suggest that the inhibition of the mTOR pathway increases sensitivity of melanoma cells towards temozolomide. Chemosensitisation is associated with enhanced expression of the Wnt antagonist DKK1.

Interstitial deletions encompassing chromosome bands 1p32.1p32.3 are rare. Only nine unrelated patients with partially overlapping 1p32.1p32.3 deletions of variable size and position have been reported to date. We report on a 17-month-old boy with choanal atresia, hearing loss, urogenital anomalies, and microcephaly in whom an interstitial de novo deletion of 6.4 Mb was detected in 1p32.1p32.3 (genomic position chr1:54,668,618-61,113,264 according to GRCh37/hg19). The deleted region harbors 31 RefSeq genes. Notable genes in the region are PCSK9, haploinsufficiency of which caused low LDL cholesterol plasma levels in the patient, and DAB1, which is a candidate gene for cognitive deficits, microcephaly, and cerebral abnormalities such as ventriculomegaly and agenesis of the corpus callosum. Choanal atresia, microcephaly, and severe hearing loss were previously not known to be associated with 1p32 deletions. Our reported patient thus broadens the spectrum of clinical findings in this chromosome region and further facilitates genotype-phenotype correlations. Additional patients with overlapping deletions and/or point mutations in genes of this region need to be identified to elucidate the role of individual genes for the complex clinical manifestations. © 2015 Wiley Periodicals, Inc.

Although immortalized cell lines have been extensively used to optimize treatment strategies in cancer, the usefulness of such in vitro systems to recapitulate primary disease is limited. Therefore, the design of in vivo models ideally utilizing patient-derived material is of critical importance. In this regard, NOD.Cg-Prkdc(scid) IL2rg(tmWjl) /Sz (NSG) mice have been reported to provide superior engraftment rates. However, limited data exist on the validity of such a model to constitute a surrogate marker for clinical parameters. We studied primary and serial engraftment on more than 200 NSG mice with 54 primary pediatric B cell precursor acute lymphatic leukemia (B-ALL), myeloid leukemia (AML) and T cell leukemia (T-ALL) samples, characterized the leukemogenic profile and correlated engraftment kinetics with clinical outcome. Median time to engraftment was 7-10 weeks and 90% of the mice engrafted. Male recipients conferred significantly higher engraftment levels than female recipients (p ≤ 0.004). PCR-based minimal residual disease marker expression and fluorescence in situ hybridization confirmed the presence of patient-specific genetic aberrations in mice. Transcriptome cluster analysis of genes known to be important in the leukemogenesis of all three diseases revealed that well-known tumor-regulating genes were expressed to a comparable extent in mice and men. The extent of engraftment and overall survival of NSG mice highly correlated with the individual prognosis of B-ALL, AML and T-ALL patients. Thus, we propose an in vivo model that provides a valuable preclinical tool to explore the heterogeneity of leukemic disease and exploit patient-tailored leukemia-targeting strategies within multivariate analyses.

Interstitial deletions of the short arm of chromosome 3 are rare. We report on a 3-year-old girl with intellectual disability, muscular hypotonia, strabismus, and facial anomalies in whom an interstitial 1.24 Mb deletion in 3p25.3-p26.1 was detected by SNP array analysis. The deleted region harbors 11 RefSeq genes including CAV3 and SRGAP3/MEGAP, which had been associated with muscle disorders and intellectual disability, respectively. The deletion overlaps with a slightly larger deletion in a girl with a more complex phenotype including congenital heart defect and epilepsy, which indicates that haploinsufficiency of one or several of the genes in the deleted interval causes intellectual deficits, but not heart defects or epilepsy. Thus, the patient broadens our knowledge of the phenotypic consequences of deletions in 3p25.3-p26.1 and facilitates genotype-phenotype correlations for chromosome aberrations of this region.

Jacobsen syndrome (JBS, OMIM 147791) is a contiguous gene syndrome caused by a terminal deletion of 11q (for review see Mattina et al., 2009). The deletion size varies between 5 and20Mb and the proximal breakpoint is localizedwithin or distal to 11q23.3. The spectrumof clinical symptoms is variable. Typical clinical signs include developmental delay (DD), preand postnatal growth retardation, thrombocytoor pancytopenia, cardiac defects, trigonocephaly and dysmorphic facies with hypertelorism, downslanting palpebral fissures, short nose with broad nasal bridge and anteverted nares, thin upper lip vermillion, V-shaped mouth, and low-set malformed ears [Grossfeld et al., 2004]. In contrast, the phenotype of a terminal duplication 16q is variable with many different and unspecific abnormalities, including intellectual disability (ID), growth retardation, CHD, gastrointestinal defects, and inconsistent facial dysmorphism [Maher et al., 1991; Savary et al., 1991; Schinzel, 2001]. We describe a 2-year-old boy with unilateral ocular coloboma and mild muscular hypotonia, who was found to have a 2.2Mb deletion of 11q25-qter and a 15.7Mb terminal duplication of 16q22.3-qter. The patient is the first child of nonconsanguineous, healthy parents. Previously, the mother had one miscarriage after 8 weeks. The child was born spontaneously following an uncomplicated pregnancy after 39 weeks of gestation with normal birth parameters (weight 3,030 g, length 54 cm, both 10th–25th centile; OFC 35 cm, 25th–50th centile). Mild muscular hypotonia and a unilateral inferior iris coloboma involving the retina and optic nerve were noted soon after birth. Cranial and renal ultrasound, echocardiogram, ultrasound examination of the hip, and hearing screening were normal. The child’s development was evaluated by basic neurological examination of the key social, motor, and language skills after birth, in the first week of life and at the age of 1, 3, 6, and 12month and 2 years by a pediatrician andnoobvious developmental delay was found. On clinical examination in our genetic clinic at an age of 6 months his weight (6,400 g, 3rd–10th centile), length (65 cm, 3rd centile), and OFC (43 cm, 10th–25th centile) were within the normal range. Apart from the iris coloboma, no other anomalies, or dysmorphic features were noted. On follow up examination at the age of 18months, themild hypotonia had ameliorated after physiotherapy and the child was able to walk without support. Figure 1 shows the boy at 19months of age. At this age he had a high forehead and low-set ears but no other dysmorphic signs. At the age of 2 years the child still had normal psychomotor development and growth parameters corresponding to the 25–50th centile (height 89 cm, weight 12 kg, head circumference 49 cm). However, by that time comprehensive developmental assessment to detect subtle developmental delay has not been performed. On repeated examinations no clinical signs for platelet dysfunction were observed. GTG-banded chromosome analysis showed a derivative chromosome 11 (Fig. 2) resulting from an unbalanced translocation: 46,XY,der(11)t(11;16)(q25;q2?3) in all 28 metaphases examined. Maternal chromosomeswere normal. The proband’s father showed a reciprocal translocation between the long arms of chromosomes 11 and 16: 46,XY,t(11;16)(q25;q2?3). Fluorescence in situ hybridization (FISH) analysis with probes from 11q25 (PAC1064E20, coordinate 130,960,053-131,065,134 bp) and 16q24.3 (PAC240G10, coordinate 88,434,421–88,552,724 bp) together with painting

This paper presents an assessment of the technical and environmental performance of a wheat-based foam (WBF) and bio-composite for shipping chilled products. The thermal conductivity of the WBF was found to be higher than that of polyurethane foams commonly used in high-value insulation packaging, but close to that of low-density (expanded polystyrene) EPS foams and significantly lower than that of polyethylene (PE) foams, which are typically used in thermal packaging of foods. The insulation performance of a simple cool box constructed from both the WBF and EPS sandwich panels without the use of any refrigerant was studied experimentally. The comparison demonstrated that the performance of the WBF cool box was comparable to that of the EPS counterpart. Two industrial case studies were conducted on WBF cool boxes with refrigerants in comparison with PE or EPS counterparts. The WBF cool boxes had comparable thermal performance to the EPS and PE counterparts on the basis of identical foam thickness. The performance of the WBF cool boxes was also simulated with finite element (FE) modelling. Good agreement was achieved between experimental data and the FE prediction. The model was then used to assist cool box design. WBF cool boxes made from renewable raw materials are inherently biodegradable and may be used as an alternative to those based on polymer foams in thermal packaging applications. Life-cycle assessment (LCA) was used to investigate environmental profiles of cool boxes made with WBF, EPS and PE foams. The WBF cool boxes offer substantially lower global warming and abiotic depletion potentials than equivalent cool boxes made from petrochemical foams. Copyright © 2010 John Wiley & Sons, Ltd.

Prenatal diagnosis of trisomy 7 is complex due to only a few reported cases. We report here on a stillborn boy with very large duplication of 7q11.22 --> qter, encompassing almost the entire long arm of chromosome 7. Ultrasound, fetal and parental chromosome banding, fluorescence in situ hybridization (FISH), and array comparative genomic hybridization (CGH) analyses were performed. Sonographic findings included growth retardation, micrognathia, ventricular septal defect (VSD), aortic coarctation, bradyarrhythmia, pericardial effusion, bilateral hydronephrosis, infravesical obstruction, and cerebellar hypoplasia. Chromosome analysis after cordocentesis at 23 weeks of gestation revealed an abnormal male karyotype with 46 chromosomes and a derivative chromosome 7 with a very large duplication of the long arm, 46,XY,der(7)(qter --> q11.2::p22 --> qter). The mother was found to carry an apparently balanced pericentric inversion, 46,XX,inv(7)(p22q11.2). Thus, the recombinant chromosome 7 [rec(7)dup(7q)inv(7)(p22.3q11.22)mat] of the fetus must have arisen through meiotic crossing-over between the inverted chromosome and the normal chromosome 7 in the maternal germline. FISH and array CGH results confirmed the recombinant chromosome 7 in the fetus and indicated a loss of 1.9 Mb at chromosome 7pter --> p22.3 (pter to 1,948,072 bp), and a gain of 87.04 Mb at chromosome 7q11.22 --> qter (71,760,154 bp to qter). The rare syndrome of almost complete trisomy 7q may be suspected in cases of growth retardation, cerebellar hypoplasia, micrognathia, aortic coarctation and VSD and hydronephrosis. Invasive prenatal diagnosis must be offered to the parents.

There are forensic inquiries in which an identification of epithelial cell types would provide important probative evidence. In cancer diagnosis, this information is yielded by histological examination of cytokeratin (Ck). Therefore, we tested 19 antibodies against different Cks (Ck1, Ck2e, Ck4, Ck5-6, Ck7, Ck8, Ck9, CK10, Ck13, Ck14, Ck15, Ck16, Ck17, Ck18, Ck19, Ck20, Ck903, PanCkAE1_3, and CAM5-2) on histological sections of epidermis, buccal mucosa, vaginal mucosa, penis, urogenital tract, and rectum and could identify two antigens unique to buccal-cell and vaginal-cell (Ck4) and skin epithelial-cell (Ck10) cytokeratin. Subsequently, we developed an immunocytological technique for distinguishing swabbed skin and mucosal cells up to at least 1 year after sampling. By the detection of the Ck4 and Ck10 mRNAs in biopsy and laser capture microdissection collected samples via quantitative real-time polymerase chain reaction, we were able to confirm our immunological findings. Hence, this study offers techniques to discriminate between skin and mucosal cells (buccal and vaginal) in forensic casework.

The Lubs X-linked mental retardation syndrome (MRXSL) is caused by small interstitial duplications at distal Xq28 including the MECP2 gene. Here we report on four novel male patients with MRXSL and different Xq28 duplications delineated by microarray-based chromosome analysis. All mothers were healthy carriers of the duplications. Consistent with an earlier report [Bauters et al. (2008); Genome Res 18: 847-858], the distal breakpoints of all four Xq28 duplications were located in regions containing low-copy repeats (LCRs; J, K, and L groups), which may facilitate chromosome breakage and reunion events. The proximal breakpoint regions did not contain known LCRs. Interestingly, we identified apparent recurrent breakage sites in the proximal and distal breakpoint regions. Two of the four patients displayed more complex rearrangements. Patient 2 was endowed with a quadruplicated segment and a small triplication within the duplication, whereas patient 3 displayed two triplicated segments within the duplication, supporting that the Fork Stalling and Template Switching (FoSTeS) model may explain a subset of the structural rearrangements in Xq28. Clinically, muscular hypertonia and contractures of large joints may present a major problem in children with MRXSL. Because injection of botulinum toxin (BT-A; Botox) proved to be extremely helpful for patient 1, we recommend consideration of Botox treatment in other patients with MRXSL and severe joint contractures.

TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

Primary torsion dystonia is an autosomal-dominantly inherited, neurodevelopmental movement disorder caused by a GAG deletion (ΔGAG) in the DYT1 gene, encoding torsinA. This mutation is responsible for approximately 70% of cases of early-onset primary torsion dystonia. The function of wildtype torsinA is still unknown, and it is unsolved how the deletion in the DYT1 gene contributes to the development of the disease. To better understand the molecular processes involved in torsinA pathology, we used genome-wide oligonucleotide microarrays to characterize gene expression patterns in the striatum of mouse models overexpressing the human wildtype and mutant torsinA. By this approach we were able to detect gene expression changes that seem to be specific for torsinA pathology. We found an impact of torsinA, independent from genotype, on vesicle trafficking, exocytosis, and neurotransmitter release in our mouse model. In addition, we were able to identify several new pathways and processes involved in the development of the nervous system that are affected by wildtype and mutant torsinA. Furthermore, we have striking evidence from our gene expression data that glutamate receptor mediated synaptic plasticity in the striatum is the affected underlying cellular process for impaired motor learning in human ΔGAG torsinA transgenic mice.

Objective: Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of neurodegenerative disorders resulting in progressive spasticity of the lower limbs. One form of autosomal recessive hereditary spastic paraplegia (ARHSP) with thin corpus callosum (TCC) was linked to chromosomal region 15q13-21 (SPG11) and associated with mutations in the spatacsin gene. We assessed the long-term course and the mutational spectrum of spatacsin-associated ARHSP with TCC. Methods: Neurological examination, cerebral magnetic resonance imaging (MRI), 18 fluorodeoxyglucose positron emission tomography (PET), nerve biopsy, linkage and mutation analysis are presented. Results: Spastic paraplegia in patients with spatacsin mutations (n 20) developed during the second decade of life. The Spastic Paraplegia Rating Scale (SPRS) showed severely compromised walking between the second and third decades of life (mean SPRS score, 30). Impaired cognitive function was associated with severe atrophy of the frontoparietal cortex, TCC, and bilateral periventricular white matter lesions. Progressive cortical and thalamic hypometabolism in the 18 fluorodeoxyglucose PET was observed. Sural nerve biopsy showed a loss of unmyelinated nerve fibers and accumulation of intraaxonal pleomorphic membranous material. Mutational analysis of spatacsin demonstrated six novel and one previously reported frameshift mutation and two novel nonsense mutations. Furthermore, we report the first two splice mutations to be associated with SPG11. Interpretation: We demonstrate that not only frameshift and nonsense mutations but also splice mutations result in SPG11. Mutations are distributed throughout the spatacsin gene and emerge as major cause for ARHSP with TCC associated with severe motor and cognitive impairment. The clinical phenotype and the ultrastructural analysis suggest a disturbed axonal transport of long projecting neurons. Ann Neurol 2007;62:656 – 665

Hepatocytes located in the periportal and perivenous zones of the liver lobule show remarkable differences in the levels and activities of various enzymes and other proteins. To analyze global gene expression patterns of periportal and perivenous hepatocytes, enriched populations of the two cell types were isolated by combined collagenase/digitonin perfusion from mouse liver and used for microarray analysis. In total, 198 genes and expressed sequences were identified that demonstrated a >/= 2-fold difference in expression between hepatocytes from the two different zones of the liver. A subset of 20 genes was additionally analyzed by real-time RT-PCR, validating the results obtained by the microarray analysis. Several of the differentially expressed genes encoded key enzymes of intermediary metabolism, including those involved in glycolysis and gluconeogenesis, fatty acid degradation, cholesterol and bile acid metabolism, amino acid degradation and ammonia utilization. In addition, several enzymes of phase I and phase II of xenobiotic metabolism were differentially expressed in periportal and perivenous hepatocytes. Our results confirm previous findings on metabolic zonation in liver, and extend our knowledge of the regulatory mechanisms at the transcriptional level.

The rate of polymerization of liquid isobutyl vinyl ether conforms to an Arrhenius temperature dependence with an overall activation energy of 6.6 kcal/ mole between --78 and 90 deg C. Also, it was found that the rate of polymerization at 30 deg C depends on the 0.65-power of the dose rate. Evidence was obtained that such results do not preclude an ionic mechanism. By studying the effect of added ion (proton) scavengers in very low concentration on the rate of polymerization, it was established that ions play the predomr-nant role as chain intermediates. The effectiveness of ammonia as a retarder for the polymerization indicates that the reaction is cationic. (P.C.H.)